Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Summary

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Abstract

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O₂ min^-1 mg total mitochondrial protein^-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Introduction

The history of plant mitochondrial research goes back over 100 years¹. Intact mitochondria were first isolated in the early 1950s using differential centrifugation. The advent of a colloidal density gradient in the 1980s allowed mitochondria to be purified without suffering osmotic adjustment. While gradient purified mitochondria are suitable for most purposes, due to the sensitivity of mass spectrometry, even relatively minor contaminants can be detected and may be inappropriately assigned a mitochondrial location². The use of free flow electrophoresis can remove both plastidic and peroxisome contamination

Protocol

This protocol is used for the isolation of intact mitochondria from Arabidopsis thaliana organs grown on soil using continuous colloidal density gradients. All procedures following the collection of the material are carried out at 4 °C.

1. Preparation of Grinding Medium, Wash Buffer, and Gradient Solutions

1. Prepare 300 mL of grinding medium (minus ascorbate and cysteine) and 200 mL of 2x wash buffer per Table 1 one day prior to the isolation, keep them cold at 4 °C.
   NOTE: Add sodium ascorbate and L-cysteine (free base) to a final concentration of 17.84 mM and 20.36 mM, respectively, to t....

Results

Using this protocol, we were able to detect different mitochondrial proteins by SDS-PAGE and immunoblotting. As shown in Figure 3A, the protein isolated from water culture tissue is sufficient to detect a faint band (2 µg). Signal intensity increases proportionately to the amounts loaded. For mitochondria isolated from tissues grown on plates (Figure 3B), the response to high light stress treatment in different genotypes can be a.......

Discussion

Typically, isolation of mitochondria from Arabidopsis leaves yields up 3 mg of mitochondria from approximately 80 - 100 3 - 4-week old plants, although yields of greater than 5 mg can often be achieved with thorough grinding. The yield varies with growth conditions and decreases dramatically as leaves senesce, although mitochondria structure seems to be well maintained during senescence⁹. One of the most critical features to obtain a good yield is the method of grinding to lyse cells to release mi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADP	Sigma-Aldrich	A2754	Chemical
Antimycin A	Sigma-Aldrich	A8674	Chemical, dissolve in ethanol
AOX antibody	from Tom Elthon		Elthon et al., 1989
Ascorbate	Sigma-Aldrich	A0157	Ascorbate Oxidase from Cucurbita sp.
ATP	Sigma-Aldrich	A26209	Chemical
Bovine serum albumin (BSA)	Bovogen	BSAS 1.0	Chemical
Clarity western ECL substrate	Bio-Rad Laboratories	1705061	Chemical
Criterion Stain-Free Precast Gels 8-16% 18 Wells	Bio-Rad Laboratories	5678104	Chemical
Cyanide	Sigma-Aldrich	60178	Chemical
Cytochrome c	Sigma-Aldrich	C3131	Chemical
Difco Agar, granulated	BD Biosciences	214530	Chemical
Dithiotreitol	Sigma-Aldrich	D0632	Chemical
EDTA disodium salt	Sigma-Aldrich	E5134	Chemical
Gamborg B-5 Basal Medium	Austratec	G398-100L	Chemical
Gamborg Vitamin Solution (1000x)	Austratec	G219-100ML	Chemical
Goat Anti-Mouse IgG (H + L)-HRP Conjugate	Bio-Rad Laboratories	1706516-2ml	Chemical
Goat Anti-Rabbit IgG (H + L)-HRP Conjugate	Bio-Rad Laboratories	1706515-2ml	Chemical
L-Cysteine	Sigma	C7352-100G	Chemical
Magnesium sulfate	Sigma-Aldrich	230391	Chemical
Murashige & Skoog Basal Salt Mixture (MS)	Austratec	M524-100L	Chemical
Myxothiazol	Sigma-Aldrich	T5580	Chemical, dissolve in ethanol
NADH	Sigma-Aldrich	N8129	Chemical
Ndufs4 antibody	from Etienne Meyer		Meyer et al., 2009
n-Propyl gallate	Sigma-Aldrich	P3130	Chemical, dissolve in ethanol
Percoll	GE Healthcare	17-0891-01	Chemical, colloidal density gradient
Polyvinylpyrrolidone (PVP40)	Sigma-Aldrich	PVP40	Chemical
Potassium cyanide	Sigma-Aldrich	60178	Chemical
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655	Chemical
Pyruvate	Sigma-Aldrich	P2256	Chemical
Sodium chloride	Chem-Supply	SA046	Chemical
Sodium dithionite	Sigma-Aldrich	157953	Chemical
Sodium L-ascorbate	Sigma	A4034-100G	Chemical
Succinate	Sigma-Aldrich	S2378	Chemical
Sucrose	Chem-Supply	SA030	Chemical
TES	Sigma-Aldrich	T1375	Chemical
Tetrasodium pyrophosphate (Na4P2O7 · 10H2O)	Sigma-Aldrich	221368	Chemical
Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit	Bio-Rad Laboratories	1704271	Chemical
Triton-X 100	Sigma-Aldrich	X100	Chemical, detergent
Western Blocking Reagent	Sigma	11921681001	Chemical
Balance	Mettler Toledo	XS204	Equipment
Beakers	Isolab	50 mL
Centrifuge	Beckman Coulter	Avanti J-26XP	Equipment
Centrifuge tubes	Nalgene	3117-9500	Equipment
Circulator	Julabo	1124971	Attached to oxygen electrode chamber
Conical flask	Isolab	500 mL
Dropper		3 mL
Fixed angle rotor	Beckman Coulter	JA25.5	Equipment
Funnel	Per Alimenti	14 cm	For filtering
Gradient pourer	Bio-Rad	165-4120	For preparation of gradients
Magnetic Stirrer ATE	VELP Scientifica	F20300165	Equipment
Miracloth	VWR	EM475855-1R	Filtration material
Mortar and pestle	Jamie Oliver	Granite, 6 Inch	Equipment
O2view	Hansatech Instruments		Oxygen monitoring software
Oxygraph Plus System	Hansatech Instruments	1187253	Clark-type oxygen electrode
Paintbrush	Artist first choice	1008R-12
Parafilm	Bemis	PM-996	plastic paraffin film
Peristaltic pump	Gilson	F155001	For preparation of gradients
PVC peristaltic tubing	Gilson	F117930	For preparation of gradients
Water bath	VELP Scientifica	OCB	Equipment